Direct Single Molecule Imaging of Enhanced Diffusion for Enzymes and Enzyme-conjugated Dna origami

Enzymes have been shown to diffuse faster in the presence of their substrates. Recently, we revealed new insights into this process of enhanced diffusion using single-particle tracking (SPT) with total internal reflection fluorescence (TIRF) microscopy. We found that the mobility of individual enzymes was enhanced three-fold in the presence of the substrate, and the motion remained Brownian. We showed that the relative increase in diffusion is independent of the total enzyme concentrations; and the oligomerization state of enzymes did not change during the catalytic turnover. These experiments ruled out the possibility that the enhanced enzyme diffusion was caused by the collective effects or the size changing of enzymes during reaction. We also compared different experimental designs with different data analysis approaches for studying single enzyme diffusion. We tried different surface coating methods (polymer brush coated surface and lipid bilayer coated surface) and different viscosity agents (methylcellulose and glycerol) to slow diffusion and facilitate tracking. We found that high amounts of glycerol inhibited enzyme activity, resulting in the failure to observe the enhanced diffusion. To get rid of glycerol, we tethered enzymes directly on the supported lipid bilayers (SLBs) and found a similar enhanced diffusion behavior for the tethered enzymes. Using active enzymes as motors, we also tried to construct highly programmable self-propelled enzyme-powered DNA origami active particles and study the mechanism of propulsion from the bottom-up